Disulfide bonds are pivotal for the correct folding, structural integrity and activity of numerous proteins found in nature. Without the correct oxidation that links their cysteines into disulfide bonds, these proteins will neither be fully stable nor biologically active. Importantly, many eukaryotic proteins of biopharmaceutical interest contain multiple disulfide bonds. Among others, these include human insulin, insulin like growth factor, human growth hormone, brain-derived neutrophic factor, nerve growth factor, lipases, Bowman-Birk protease inhibitor, and antibody fragments.
The formation of disulfide bonds can occur spontaneously, but this process is very slow and non-specific. For this reason, enzymes have evolved that catalyze the formation (oxidation) of disulfide bonds in vivo. These enzymes belong to the class of thiol-disulfide oxidoreductases (TDORs). This class of enzymes also contains enzymes that break (reduce) or isomerise disulfide bonds. Cytoplasmic TDORs generally function as reductases while their extracytoplasmic equivalents are oxidases or isomerases (Dorenbos et al., (2005) p. 237-269. In S. G. Pandalai (ed.), Recent Res. Devel. Microbiology 9. Research Signpost, Kerala, India; Ritz et al., Annu. Rev. Microbiol. 55:21-48, 2001; Tan et al., Chembiochem. 5:1479-1487, 2004). The enzyme-dependent formation of disulfide bonds is, in fact, a prime reason why proteins containing such bonds are still troublesome to produce in large amounts using bacterial cell factories. Slow and/or non-specific oxidation of overproduced proteins in bacterial cell factories may result in slow and/or incorrect folding of these proteins, making them vulnerable to proteolytic degradation and potentially rendering them inactive unless they are further processed in vitro into correctly folded and active product.
Previous studies on disulfide bond formation in Bacillius subtilis have shown that this organism contains at least four TDORs with presumed oxidase activity. These proteins were named Bdb (Bacillus disulfide bond) proteins, and annotated as BdbA-D. The bdbA and bdbB genes are located within the SPβ prophage region, and are therefore only present in the sequenced B. subtilis strain 168. Biological functions have been identified for BdbB, BdbC and BdbD, but not for BdbA. The Bdb function was found to be modular in the sense that different Bdb proteins can cooperate to perform different functions (Kouwen et al., Mol. Microbiol. 64:984-999). The integral membrane protein BdbB shares a high degree of sequence similarity with BdbC and both are of major importance for folding of the secreted SPβ-encoded lantibiotic sublancin 168, which contains two disulfide bonds (Bolhuis, et al., J. Biol. Chem. 274:24531-24538, 1999, Dorenbos et al., J. Biol. Chem. 277:16682-16688, 2002, Stein, Mol. Microbiol. 56:845-857, 2005). On the contrary, BdbC together with BdbD are of major importance for the biogenesis of the pseudopilin ComGC, while BdbB is dispensable for this process (Meima, J. Biol. Chem. 277:6994-7001, 2002). ComGC is an important element of the DNA-uptake machinery of B. subtilis and, consistent with its TDOR requirement for folding into a protease-resistant conformation, it contains an essential intra-molecular disulfide bond (Chung et al., Mol. Microbiol. 29:905-913, 1998). BdbC and BdbD are also required for folding of a secreted heterologous protein by B. subtilis, namely the alkaline phosphatase PhoA of E. coli (Bolhuis et al., J. Biol. Chem. 274:24531-24538, 1999; Darmon et al., Appl. Environ. Microbiol. 72:6876-6885, 2006, Kouwen et al., Mol. Microbiol. 64:984-999. 2007, Meima et al., J. Biol. Chem. 277:6994-7001, 2002). This TDOR requirement relates to the fact that E. coli PhoA contains two disulfide bonds that are indispensable both for the enzymatic activity and stability of this protein (Sone et al., J. Biol. Chem. 272:6174-6178, 1997). Taken together, these previous observations indicate that the combined BdbA-D proteins provide the basic machinery for the folding of both homologous and heterologous disulfide bond-containing proteins in B. subtilis. 
Bacillus organisms also contain thioredoxins, which are small, heat stable, ubiquitous TDORs that are involved in a large variety of processes, ranging from enzyme activation to mitochondria-dependent apoptosis (Tanaka et al., EMBO J. 21:1695-1703, 2002). During catalysis, the cysteine residues of their CxxC active site undergo a reversible oxidation-reduction reaction. In the bacterial cytoplasm, thioredoxin is usually present in a reduced state in order to prevent the formation of disulfide bonds in cytoplasmic proteins.
It has been reported that BdbC and BdbD cooperate as a redox pair in an oxidation pathway of B. subtilis (Sarvas et al., Biochim. Biophys. Acta 1694:311-327, 2004). It was therefore proposed that BdbD functions as the major oxidase for secreted cysteine-containing proteins, thereby facilitating the formation of disulfide bonds. Subsequently, the reduced BdbD would be re-oxidized by the quinone reductase homologue BdbC. To become re-oxidized for a next catalytic reaction, BdbC would then donate its electrons to quinones in the electron transport chain. This system resembles the DsbA and DsbB redox pair of E. coli (Inaba et al. Cell 127:789-801, 2006), Regeimbal et al. J. Biol. Chem. 277:32706-32713, 2002), Rietsch & Beckwith. Annu. Rev. Genet. 32:163-184, 1998). Despite the presence of four Bdb proteins, the total oxidative power of B. subtilis is rather limited. In an attempt to increase the thiol-oxidizing capacity, overexpression of individual or combinations of Bdb proteins has been attempted. However, this did not result in significantly improved production of proteins with disulfide bonds (Darmon et al., Appl. Environ. Microbiol. 72:6876-6885, 2006, Dorenbos et al., J. Biol. Chem. 277:16682-16688, 2002, Meima et al., J. Biol. Chem. 277:6994-7001, 2002).
There have been other reports that host cells, such as bacteria, exhibit relatively poor performance in the production of proteins with disulfide bonds (Braun et al., Curr. Opin. Biotechnol. 10:376-381, 1999; Anfinsen, Science 181:223-230, 1973; Westers et al., Biochim. Biophys. Acta 1694:299-310, 2004).